Estrogen binding sites in the hypothalamus-preoptic area (HPOA) of adult male and female gray opossums (Monodelphis domestica) were characterized by incubating cytosols from gonadectomized animals with [3H]estradiol (E2) in the presence or absence of excess unlabeled E2. Scatchard analyses revealed that HPOA cytosols from both males and females contained high concentrations (25-30 fmol/mg protein) of binding sites with a very high affinity (Kd = 0.08-0.12 nM) for E2. There was no sex difference in either the number or the affinity of estrogen binding sites. Competition studies indicated that estrogen agonists and antagonists displaced [3H]E2 from binding sites in HPOA cytosols more effectively than did progestins, androgens, or adrenal corticoids. These data suggest that the HPOA of both male and female gray opossums contains macromolecules with several of the steroid binding properties characteristic of estrogen receptors.